To describe the mechanism by which E2F regulates dhfr transcription, the amplified dhfr gene of CH0C 400 cells will be used as endogenous reporter genes for E2F activity. CH0C 400 cell lines that conditionally express E2F, DP, or RB family member proteins will be used to relate the formation of specific E2F/DP/pRB family protein complexes to: 1) dhfr gene transcription as measured by nuclear run-on assays, 2) dhfr mRNA levels, 3) the genomic footprint of the dhfr promoter, and 4) progression through the cell cycle. Genomic footprinting in synchronized cells also will be used to gain information regarding the order of assembly of transcription factors on the dhfr promoter after DNA replication. In vitro DNA binding studies with recombinant E2F/DP complexes and defined DNA substrates will be used to study the conserved architecture of the overlapping, inverted E2F sites at the dhfr promoter. Sequence alterations that influence E2F/DP DNA binding in vitro will be tested for transcriptional regulation in vivo. Promoter reconstruction and expression of mutant E2F, SP and pRB family member proteins will be used to test models for dhfr gene expression in whole cells. In related work, it has been shown that DP-1 is ubiquitinated when expressed at high levels in CH0 and human cells. Regions of DP-1 required for ubiquitination will be mapped, and the role of ubiquitination in DP-1 function will be examined. These studies will provide new information concerning the mechanism by which E2F, DP and pRB family members act in concert to regulate transcription of an endogenous cellular gene during the cell cycle.